Next Generation Sequencing (NGS, deep sequencing) is revolutionizing the use of sequence information to understand genomes and genome variability. While NGS has allowed the rapid sequencing of large genomes, it has also allowed the allowed an analysis of the genetic diversity in organisms with small genomes. However, the use of NGS to measure population diversity is fraught with artifacts in when each sequence is considered as an independent observation/genome. The high error rate of the sequencing protocols and misincorporation during PCR amplification introduce artifactual diversity. PCR resampling of the same template sequences after amplification introduces artifactual homogeneity. Neither of these serious problems is addressed in most of the application of NGS to complex populations such as HIV-1. We have developed the Primer ID approach specifically to overcome these problems. In this application we propose to define the strengths and weaknesses of the Primer ID strategy and to use this strategy to explore fundamental features of HIV-1 population structure and evolution. These studies will provide definitive answers to longstanding and important questions that impact the relevance of pre-existing variants prior to drug therapy and the potential pathways for HIV-1 evolution. The experiments will use human samples so that the results will be directly applicable to understanding the nature of virus-host interactions in the context of this pathogenic infection.